The present invention relates to cloning vectors for use in Rhodococcus and related bacteria.
Members of the genus Rhodococcus are gram-positive, aerobic, non-sporulating, partially acid-fast Actinomycetes, which were formerly classified as Nocardia, Mycobacterium, Gordona, Jensenia, or in the "rhodochrous" complex. Nocardia, Corynebacteria and Mycobacterium are closely related to Rhodococcus, each exhibiting nocardioform morphology, having mycolic acids, meso-diaminopimelic acid, arabinose and galactose in their cell walls and having a high G+C content (&gt;59 mol %) in their cellular DNA. Most members of the genus are saprophytic soil organisms, although several pathogenic species exist, including R. bronchialis, a human pathogen, R. equi, an animal pathogen and R. fascians, a plant pathogen.
Rhodococci exhibit a wide range of metabolic activities including antibiotic production, amino acid production, degradation of alkanes and aromatic hydrocarbons, biotransformation of steroids and a number of xenobiotic compounds, lignin degradation, chemolithoautotrophic growth in the presence of hydrogen and carbon dioxide and production of biosurfactants.
Genetic studies in Rhodococcus have focused on mapping the R. erythropolis chromosome, with approximately 65 chromosomal markers established, using a natural mating and recombination system, as reported by Brownell et al., The Biology of the Actinomycetes, M. Goodfellow, eds., pp. 201-228 (Academic Press, N.Y. 1984). A lysogenic actinophage, .phi.EC, a 47 Kb double stranded DNA phage, has been physically mapped by restriction analysis for potential use as a cloning vector in Rhodococcus, as described by Brownell, et al., Gene 12, 311-314 (1980) and Dev. Ind. Microbiol. 23, 287-298 (1982). Phage .phi.EC can be transferred between fertile Rhodococcus strains either as a plasmid or as a prophage and phage .phi.EC DNA can be transfected into R. erythropolis protoplasts. Native plasmids have been described by Reh in Zbl. Bakt. Suppl. 11, 577-583 (1981) and by Sensfuss, et al., in J. Gen. Microbiol. 132, 997-1007 (1986) in the hydrogen-oxidizing autotrophic strain Rhodococcus sp. (Nocardia opaca 1b). The self-transmissible trait, Aut+, encoding genes for autotrophic growth in this strain, was previously thought to be plasmid-localized, but is now considered to be a chromosomal trait. Thallium resistance is associated with large plasmids, (110-140 Kb) in two Aut+ strains described by Sensfuss, et al.
Although cloning vectors have been reported for a variety of Gram negative organisms, especially E. coli, and a few Gram positive organisms such as Corynebacterium and Bacillus, to date, no one has provided a vector which can be used to transfer foreign DNA into Rhodococcus for transcription and translation into protein.
It is therefore an object of the present invention to provide a cloning vector for transforming DNA into Rhodococcus.
It is another object of the present invention to provide a cloning vector which can be used as a shuttle vector between Rhodococcus and E. coli and other bacteria.
It is a further object of the present invention to provide a method and means for constructing additional cloning vectors for use in Rhodococcus and related bacteria.